The development of immunological techniques for the detection of disease associated materials such as antigens by their immunological reactivity with substances specific therefor, typically called antibodies, has been hindered by a number of technical problems. In particular, one of these problems is the so-called hook effect which introduces significant nonlinearity in assay sensitivity at high antigen concentrations. An additional undesireable characteristic is the accompanying limit in dynamic range.
The hook effect is perhaps most easily understood in relation to the standard sandwich assay for the detection of a ligand. Typically, such an assay employs a first ligand binding partner, insolubilized such as by attachment to the wall of a microtitration tray well, and a second ligand binding partner having associated therewith a detectable label. Mixing together an aqueous body fluid sample suspected of containing the ligand to be detected, along with the aqueous labeled second binding partner and the immobilized first binding partner under suitable conditions, e.g. incubation at 37.degree. C. for sufficient time, permits the formation of a sandwich complex wherein the first binding partner specifically reacts with the ligand to be detected, this results in its immobilization onto the wall of the micotitration well. Similarly, the second binding partner reacts with the ligand and thus the label associated therewith also becomes immobilized. Soluble components remaining in solution which have not reacted are thereafter removed and the amount of label in either the soluble or insoluble phase detected. The amount of label detected can be related to the quantity of ligand present in the fluid sample.
Typically, the immobilized first ligand binding partner is supplied in excess in order to ensure that sufficient binding sites are available for the retention of all possible ligands within the sample. The quantity of second ligand binding partner supplied in the reagent will, of course, be fixed and is generally set at a concentration level well below that which would result in nonspecific absorption to the insoluble phase, but still sufficiently high to thereby provide enough label for attachment to a reasonably high, clinically significant ligand concentration.
Unfortunately, however, patient samples are often not so accommodating and may present a far wider range of concentrations of ligands requiring a greater dynamic range of sensitivity response than such an assay has heretofore afforded. In particular, the hook effect becomes significant with very large ligand concentrations. In such situations, there is so much ligand present in the sample that all available combining sites on the immobilized first ligand binding partner as well as those available on the second labeled ligand binding partner are filled with the available ligand. Indeed, there may still be additional unattached ligands available. As a result of the plethora of ligands available fewer sandwich complexes are being formed since only some of the first and second ligand binding partners will be attached to the same ligand. Consequently, an increasing ligand concentration results in a proportional increase in immobilized label until the ligand concentration becomes so great that fewer sandwich complexes are formed whereupon the curve rapidly drops off giving a false, lower concentration of ligand. This is the hook effect.
It will be readily appreciated that the predominant danger introduced by the hook effect is the erroneous indication of far smaller ligand concentrations than are actually present in the sample. Conventional immunoassay methods have attempted to address the hook-effect problem by supplying greater numbers of both the labeled and immobilized ligand binding partners in order to accommodate greater ligand concentrations. This approach, however, disadvantageously results in greater economic costs and is further constrained by present technology which limits the amount of ligand binding partners which may be immobilized per unit area on a solid phase. Such increased concentrations may also be at the expense of sensitivity since increasing the numbers of binding partners and the area available for attachment of binding partners can result in greater difficulty in locating and forming sandwich complexes with the few ligands that may be present in a sample having a very low ligand concentration.
It is one aspect of the present invention to reduce the hook effect in clinically useful ligand concentration ranges without increasing the immobilized or labeled ligand binding partners.
The other conventional alternative for addressing the hook-effect practiced to date is serial dilution of samples having ligand concentrations at some preset level in order to bring the sample within the dynamic range which may be handled by the assay system. This disadvantageously results in additional steps; indeed a complete duplication of the assay for those samples having high ligand concentrations.
It is yet another aspect of the present invention to reduce the number of occasions when dilution of patient samples is required by extending the dynamic range of sensitivity.
Related copending, commonly assigned, U.S. application Ser. No. 722,110 entitled "Immunoassay Methods for Single and Multiple Epitopic Ligands Utilizing Isotactic Surfaces" and fully incorporated herein by reference describes an immunoassay system for the detection of a variety of ligands utilizing a standard procedure and standard volumes of samples and reactants.
It is still yet another aspect of the present invention to provide a method for reducing the hook-effect whereby the volumes of the reactants in the immunoassay system described in U.S. Ser. No. 722,110 may be standardized and the need to perform serial dilutions is reduced due to the extension of sensitivity dynamic range.